Transcription is the first step in gene expression and thus is highly regulated. Transcription is carried out by multi-subunit RNA polymerases (RNAPs) that are conserved from bacteria to humans. Therefore, insight into the mechanism and regulation of the bacterial RNAP provides insight into the mechanism and regulation of RNAPs in all cells. The proposed studies are part of a long-range effort to obtain a quantitative understanding of transcription that confers the ability to predict transcriptional output from the DNA sequence content of a gene. Towards achievement of this goal, the proposed studies will apply sophisticated high-throughput sequencing methodologies to systematically define the relationship between nucleic acid sequence and functional output during transcription initiation for bacterial RNAP. The first aim will systematically define the relationship between the sequence of the promoter DNA and sensitivity to DNA topology (supercoiling) during transcription initiation. The second aim will define the functional role of sequence-specific interactions between RNAP and the core recognition element, CRE during transcription initiation. The third aim will comprehensively document the impact of sequence in the process of reiterative transcription initiation. The fourth aim will systematically document the stabilities o transcription complexes as a function of promoter sequence and establish whether or not the stabilities of each promoter variant can allow prediction of the sensitivity to regulatory factors ppGpp and DksA.